Project Summary/Abstract Fifty percent of A. baumannii isolates from US ICUs are extreme drug resistant (XDR), far higher than for other pathogens. These infections result in ?10,000 and 30,000 deaths and excess healthcare costs of $390 million and $742 million in the US and globally, annually. Furthermore, in contrast to other resistant bacteria, virtually no antibiotics are in the pipeline to deal with XDR A. baumannii. New treatments are critically needed. We established two MAbs, C8 and 39, that collectively bind to 90% of the 62 clinical isolates of A. baumannii tested. We have found efficacy in lethal murine models of XDR A. baumannii bacteremic sepsis and aspiration pneumonia, the two most common sites of A. baumannii infection. Furthermore, C8 exhibits substantial synergy with colistin during delayed therapy for lethal A. baumannii infection (39 is not yet tested). Other MAbs have also been raised that are not yet characterized. We will define an optimal mixture of MAbs to target strains broadly, and define mechanisms of protection to support future humanization and clinical trials. Specific Aim 1: Define optimal MAbs with respect to surface binding and in vitro killing of multiple clinical isolates of A. baumannii. We will define surface binding of the MAbs vs. isotype controls to broadly diverse clinical isolates of A. baumannii. We will also identify the mechanism of bacterial clearance by the MAbs, the potential for and frequency of bacterial escape mutants, and the impact of MAbs on antibiotic susceptibility. Finally, we will establish the antigenic targets of the MAbs. Specific Aim 2: Define the in vivo effects of the optimal MAbs in 3 models of infection and against multiple strains of A. baumannii, with and without antibiotics. We will compare in vivo efficacy of single vs. combination MAbs vs. isotype control during delayed therapy in 3 well-established models of infection; mouse bacteremic sepsis, mouse pneumonia, and rat wound infection. We will evauate for anti-MAb antibodies in rats. We will then determine how early prior to infection MAb prophylaxis is effective, and how long post-infection MAb therapy remains effective, and the impact of multiple doses of MAbs during prolonged therapy. Finally, we will define interactions between MAbs and antibacterial therapy in each model. Specific Aim 3: Define the mechanisms of protection of optimal MAb passive vaccination. We will define the in vivo mechanisms of efficacy by treating with optimal MAb vs. isotype control in wild-type vs. mice with selective depletion of host effectors (e.g., complement, macrophages, neutrophils, and activating Fc?R vs. inhibitory Fc?RII). Outcome measures will include survival, bacterial density, and inflammation. These results will inform future efforts to optimize the efficacy of humanized MAbs, and identify surrogate efficacy assays. Novel solutions for A. baumannii infections are a critical unmet need. We will identify an optimal MAb regimen protective across multiple sites of infection (blood, lung, soft tissue), define in vitro correlates of protection, and determine the mechanisms of protection, which will enable future efficacy optimization of humanized MAbs.